The phase coherence of the neurovascular unit is reduced in Huntington’s disease

Abstract Huntington’s disease is a neurodegenerative disorder in which neuronal death leads to chorea and cognitive decline. Individuals with ≥40 cytosine–adenine–guanine repeats on the interesting transcript 15 gene develop Huntington’s disease due to a mutated huntingtin protein. While the associated structural and molecular changes are well characterized, the alterations in neurovascular function that lead to the symptoms are not yet fully understood. Recently, the neurovascular unit has gained attention as a key player in neurodegenerative diseases. The mutant huntingtin protein is known to be present in the major parts of the neurovascular unit in individuals with Huntington’s disease. However, a non-invasive assessment of neurovascular unit function in Huntington’s disease has not yet been performed. Here, we investigate neurovascular interactions in presymptomatic (N = 13) and symptomatic (N = 15) Huntington’s disease participants compared to healthy controls (N = 36). To assess the dynamics of oxygen transport to the brain, functional near-infrared spectroscopy, ECG and respiration effort were recorded. Simultaneously, neuronal activity was assessed using EEG. The resultant time series were analysed using methods for discerning time-resolved multiscale dynamics, such as wavelet transform power and wavelet phase coherence. Neurovascular phase coherence in the interval around 0.1 Hz is significantly reduced in both Huntington’s disease groups. The presymptomatic Huntington’s disease group has a lower power of oxygenation oscillations compared to controls. The spatial coherence of the oxygenation oscillations is lower in the symptomatic Huntington’s disease group compared to the controls. The EEG phase coherence, especially in the α band, is reduced in both Huntington’s disease groups and, to a significantly greater extent, in the symptomatic group. Our results show a reduced efficiency of the neurovascular unit in Huntington’s disease both in the presymptomatic and symptomatic stages of the disease. The vasculature is already significantly impaired in the presymptomatic stage of the disease, resulting in reduced cerebral blood flow control. The results indicate vascular remodelling, which is most likely a compensatory mechanism. In contrast, the declines in α and γ coherence indicate a gradual deterioration of neuronal activity. The results raise the question of whether functional changes in the vasculature precede the functional changes in neuronal activity, which requires further investigation. The observation of altered dynamics paves the way for a simple method to monitor the progression of Huntington’s disease non-invasively and evaluate the efficacy of treatments.


Introduction
This document contains supplementary material for the paper "The phase coherence of the neurovascular unit is reduced in Huntington's disease".The lay-out of the fNIRS and EEG probes used to record data discussed in the paper is shown in Supplementary Figure 1.
Supplementary Figure 1: EEG electrode and fNIRS channel placements.Note that in 8 locations EEG and fNIRS probes are co-located.
2 Effect sizes 2.1 The effect size that can be reliably detected The data for this study were collected in April and May 2018.The effect size sensitivity was calculated based on a power of 0.8, a significance level of 0.05 and considering the existing sample sizes.We then found the effect size that the study can reliably pick up.It is 1.03 for the pre-symptomatic HD and 0.96 for the symptomatic HD, which means the study can reliably find large differences between the groups.The calculations were done using G*Power [3], and an example is shown in Supplementary Figure 2. Supplementary Figure 2: Screenshot from G*Power, showing the calculation of the effect size that this study can reliably detect in the case of the S vs. SC comparisons.S = symptomatic HD, SC = control group for S.

Evaluation of the effect size
The effect size was calculated post-hoc using Cohen's d with a non-parametric adjustment [4,5].The z-value is the standard score, calculated from the ranks when applying the Wilcoxon rank-sum test [6].Based on the z-value, r is calculated as where N is the number of participants [6].Cohen's d is found as A Cohen's d greater than 0.8 is considered large, while a value between 0.5 and 0.8 is considered medium.The results are summarised in Supplementary Table 1.The effect size is calculated for cases where p ≤ 0.05 was obtained, so that the differences are statistically significant.For the coherence and alpha power, we show the minimum, maximum and mean effect size of all significant combinations.The results are summarised in Supplementary Table 1, and a large effect size obtained from the nonparametric Cohen's d is indicated in red, while a medium effect size is indicated in blue.
Supplementary Table 1: Effect size for the significant parameters, using the parametric Cohen's d (refered to as Cohen's d), nonparametric Cohen's d, and r.For the fNIRS power the effect size is calculated for each probe where a significant difference is found.The information is given as probe: effect size.For coherence and alpha power the effect size is calculated for the significant combinations/electrodes, and we show the mean, minimum and maximum values.Positive values mean that the control groups had higher values than the HD groups, while negative values mean the opposite.For the nonparametric Cohen's d, large effect sizes are indicated in red and medium effect sizes in blue.S = symptomatic HD, SC = control group for S, P = presymptomatic HD, PC = control group for P.
For the fNIRS, EEG and fNIRS-EEG power/coherence calculations, significant differences were assessed also using a Monte-Carlo permutation test.Participants from the P and CP groups were randomly placed into two groups of size 13 and 29, while participants from the S and SC groups were randomly placed into two groups of size 15 and 33.The Wilcoxon rank-sum test was then applied to test for differences between these groups.After ∼ 16000 permutations of the groups, the original p-value was compared to the new ones.Supplementary Figure 3 shows an example.95.1% of the randomly found p-values were above 0.05, and the original p-value was smaller than 98.7% of them.Another example is shown in Supplementary Figure 4. 95.2% of the randomly found p-values were above 0.05, and the original p-value was smaller than 97.5% of them.Supplementary Figure 3: (A

Reproducibility
The number of participants is a limiting factor in our study, which raises the important question of the reproducibility of the results obtained.Due to the intensive nature of the experimental protocol further measurements are not possible at this time.We have therefore addressed reproducibility in the following ways: • Where appropriate we have compared the first and last 10 minutes of recordings, to assess the consistency of the differences between the groups throughout the session.We did this for the fNIRS and EEG power, but not for the coherence calculations as the latter comparisons require longer recordings.The results of the power comparisons can be seen in Supplementary Figures 5 and 6.
• When comparing the coherence between groups, only the effective coherence was considered.The effective coherence is found by subtracting the 95th percentile of the surrogate coherences found at each frequency, creating a high surrogate threshold [7,8].This ensures that the coherence is statistically significant.
• We have used long recordings (20 minutes).The properties analysed must be present over sufficient time for significant differences between groups to be detected: sporadic, random changes would average out over time in such long recordings.
Supplementary Figure 5: Significant p-values, found using the Wilcoxon rank-sum test, for oxyHb power for the first 10 minutes of the signals and the last 10 minutes of the signals.The first rows are between the P and PC groups, while the second rows are between the S and SC groups.Yellow/red (blue/purple) circles indicate that the power is higher in the controls (HDs).P = presymptomatic Huntington's disease, S = symptomatic Huntington's disease, PC = control group for P, SC = control group for S.
) Histogram of the p-values obtained for fNIRS-EEG coherence in the myogenic band at EEG P8 and fNIRS 10, for the S and SC groups.The red line is the original p-value.There are 1000 bins in the histogram.(B) Zoom of the smaller p-values in (A).Supplementary Figure 4: (A) Histogram of the p-values obtained for fNIRS myogenic power at probe 1 for the P and PC groups.The red line is the original p-value.There are 1000 bins in the histogram.(B) A zoom of the smaller p-values shown in (A),